This invention relates to capillary electrophoresis, or as it is more conventionally called "capillary zone electrophoresis" (CZE), and more particularly to automated methods and apparatus for introducing samples into capillary columns and for improving separations by using temperature control of said columns.
In recent years significant advances have been made in micro-column separation techniques. A principal advantage of such techniques is their suitability for analysis of extremely small sample volumes, e.g. in the microliter or submicroliter amounts of sample. Beino able to analyze such small volumes has become exceedingly important with the explosion of research in the biological field, because often-times biological samples are quite small.
One of the significant problems with capillary techniques is in introducing sample into the capillary. One technique used in capillary electrophoresis, called sample injection, is electromigration, a term collectively including the effects of eletrophoresis and electro-osmosis (See Jorgenson, J. W, and Lukacs, K. D., J. Chromatography, 1981, Vol. 218, pp. 209-216; Jorgenson, J. W., and Lukacs. K. D., Science, 1983, Vol. 222, pp. 266-272; and Wallingford, R. A. and Ewing, A. G., Anal. Chem., 1987, Vol. 59, pp. 681-684). In this technique, one end of the capillary and the electrophoresis anode are placed into the sample and a voltage is briefly applied, causing a small band of sample to electromigrate into the capillary. This method of sample injection suffers from discrimination within the sample because solutes with higher mobilities will preferentially migrate into the electrophoresis column and therefore change the relative composition of the sample. To avoid this problem, attempts to physically inject sample have also been reported (Jorgenson and Lukacs, Science, ibid). However these direct injection techniques cause band broadening, apparently due to the laminar flow profile introduced during the injection.
Other less common injection methods include gravity flow (See Tsuda, A., et al, J. Chromatography, 1983, Vol. 264, pp. 385-392.), siphoning (See Honda, S. et al, J. Chromatography, 1987, Vol. 404, pp. 313-320.), and the use of an electonic sample splitter (See Deml, M. et al, J. Chromatography, 1985, Vol. 320, pp. 159-165.). Each of these injection techniques are capable of placing subnanoliter volumes of sample into the electrophoresis column with a minimum of band broadening. However, the gravity flow or siphoning injection method is inaccurate and lacks precision in providing absolute volume amounts due to the unreliable position of the sample level which will change due to the sample withdrawal. The latter can only be neglected if the original sample volume is large compared to the volume injected. With the electronic splitter, a larger initial sample volume is required in order be able to split it down to the smaller size required for the column. Thus, some sample may be wasted, or there may not be sufficient sample to perform a separation. Also, this latter technique is further complicated by requiring an additional controlled power supply or very careful control of the electric resistances in the different legs of the splitter. Furthermore, the need to use an initial larger sample size significantly decreases the number of applications for which it can be used.
What is needed is a simple, automatable, sample injection technique that is suitable for microvolumes, is capable of providing accurate sample volumes, and which produces a minimum of band broadening.